Plasma contains one of the most complex and useful human proteomes. Detection of proteins within this type of sample is currently an important tool for evaluating the predisposition, presence, and progression of numerous clinical conditions. However, the current methodology of detecting and measuring individual proteins only begins to scratch the surface of its full potential. Today, tests for only about 120 different protein analytes have been approved by the FDA and approval of tests to detect new protein analytes has averaged only about one test per year over the last decade. This represents only an extremely small fraction of the 40,000 different proteins present in normal plasma and another 500,000 proteins which may be present under a variety of clinical conditions. This has led investigators to attempt to develop methods using high-throughput detection methods to identify many more proteins in the plasma. Current methodology, based on 2D gels, liquid chromatography, and/or mass spectrometry, has lead to the detection of about 500 different plasma proteins. However, this appears to be the limit of detection for the analysis of unfractionated plasma due to the limited dynamic range of these analytical methods. Abundant proteins, such as albumin (35-45 mg/ml), fibrinogen (2-6 mg/ml), immunoglobulins (12-18 mg/ml), and transferrin (2-3 mg/ml), interfere with the detection of proteins that may be present at up to 10 orders of magnitude lower concentrations. Removal of the ten most abundant proteins only increases the sensitivity by one order of magnitude, still leaving most proteins undetected. This has led some to attempts to fractionate the plasma samples to reduce its complexity prior to analysis.
Microparticles (MPs) are small subcellular membranous vesicles released by essentially all cell types, especially when activated or under stress. They include ectosomes, generated from the ectocytosis (or blebbing) of the plasma membrane, and exosomes, released by fusion of intracellular multivesicular endosomes with the cell surface. In plasma, MPs were discovered as a blood component that promotes coagulation due to the presence of anionic phospholipids on their outer surface. These anionic phospholipids, which are mostly phosphatidylserine, are now widely used to detect MPs from blood samples using flow cytometry based on their affinity for fluorescently labeled annexin V. The cellular source of microparticles can be determined by cell-specific markers detected by flow cytometry. For example, MPs with CD41 (Glycoprotein IIb) expression are believed to be generated by platelets. Using this method, microparticles from platelets, erythrocytes, endothelial cells, neutrophils, lymphocytes and even smooth muscle cells have been detected in plasma. In healthy individuals, over 90% of these MPs originate from platelets, and under a wide range of pathological conditions the total number of microparticles and the number originating from various cell population are altered.
Traditionally, the presence of these microparticles in the plasma is considered a sign of cellular activation and/or damage generated by the random blebbing of cell membranes. However, significant evidence has accumulated to indicate that MPs contain a unique set of proteins and inflammatory factors that have important biological functions. For example, a major pathway for the rapid secretion of IL-1β from THP-1 monocytes occurs via MP release in ectosomes, and this could be a general mechanism of release for secreted proteins that lack the conventional signaling peptide for secretory proteins. Researchers using MPs isolated either from plasma or the supernatants of stimulated cells have demonstrated a wide variety of effects on cellular function. These effects of MPs include increased expression of adhesion molecules on endothelial cells and monocytes, stimulation of cytokine release, altering vascular reactivity, inducing angiogenesis, decreasing the response to inflammatory mediators, and promoting fibrin formation. The effects vary depending on the cellular source of the microparticles, the method to generate them and the cells (or tissue) affected. While many groups have studied the effects of these MPs on various cells, the protein composition of plasma MPs was largely unknown except for the presence of a few cell-surface markers. Recently, we analyzed the proteome of platelet-derived microparticles and identified 578 proteins. Many microparticles in plasma are derived from platelets, but it is believed that valuable information resides in plasma microparticles derived from other cells as well.
It is therefore desirable to find methods of identifying biomarkers in plasma microparticles that can be useful for diagnosing various diseases, disorders, and conditions.